1. Field of the Invention
This invention relates generally to the pharmacological treatment of hepatitis C virus infection in patients.
2. Description of the Related Art
Hepatitis C virus (HCV), the putative agent in the majority of post-transfusion acquired hepatitis, has been recently defined by a new serologic assay. Kuo, G., et al., Science, 244:362-4 (1989). Despite improvement in the quality of the blood-donor pool and the recent implementation of testing of donated blood, the current estimated incidence of acute infection among persons receiving transfusions is 5 to 10%. Alter, H. J., in Zuckerman, A. J., ed., Viral Hepatitis and Liver Disease, Allen K. Liss, New York, 1988, pp. 537-42. Chronic hepatitis develops in at least half the patients with acute HCV infection (representing about 90% of patients with non-A, non-B hepatitis (NANB)), and cirrhosis develops in at least 20% of this group. Thus, of the approximately 3 million persons who receive transfusions in the United States each year, acute hepatitis C will develop in about 150,000. Chronic hepatitis C will develop in at least 75,000 of these, and among them cirrhosis will develop in more than 15,000. Among patients with post-transfusion hepatitis, up to about 90% are positive for the HCV antibody. Davis, G. L., et al., New England Journal of Medicine, 321:1501-6 (1989). Patients with sporadic NANB hepatitis (no specific risk factors) are also very likely to have the anti-HCV antibody. Kuo, et al. (1989) above. While most of the patients who contract hepatitis C will have subclinical or mild disease, approximately 50% will progress to a chronic disease state characterized by fluctuating serum transaminase abnormalities and inflammatory lesions on liver biopsy. By some estimates, cirrhosis will develop in up to about 20% of this group. Koretz, R. L., et al., Gastroenterology, 88:1251-4 (1985).
With the aim of halting or slowing the progression of HCV-related diseases, a variety of drugs have been evaluated in recent years. Both acyclovir and corticosteroids (which are beneficial in autoimmune chronic active hepatitis) are ineffective. Pappas, S. C., J. Med. Virol., 15:1-9 (1985); Stokes, P., et al., Gastroenterology, 92:1783 abstract (1987).
To date, .alpha.-interferon (IFA) appears to be the most promising candidate, although not necessarily the final answer. Hoofnagle, J. H., et al., in Viral Hepatitis: 1981 International Symposium, Philadelphia, Franklin Institute Press, 1982, pp. 573-83; Hoofnagle, J. H., et al., New England Journal of Medicine, 315:1575-8 (1986); Thomson, J., Lancet, 1:539-41 (1987); Kiyosawa, K., et al., in Zuckerman, A., ed., Viral Hepatitis and Liver Disease, Allen K. Liss, New York, 1983, pp. 895-7. Hoofnagle, J. H., et al., Sem. Liver Dis., 9:259-263 (1985). The interferons are host proteins made in response to viral infections as well as other antigenic stimuli. They are classified by their cell of origin as well as their antigenicity. .alpha.-Interferon is made by lymphoblastoid cells, .beta.-interferon by fibroblasts, and .gamma.-interferon by T-cells. Subtypes in each group are based on antigenic/structural characteristics. Recombinant forms for each group have been developed and are commercially available. A pilot study utilizing IFA on ten patients with well-characterized post-transfusion NANB hepatitis was reported in 1986 by Hoofnagle et al. (Hoofnagle, J. H., et al., New England Journal of Medicine, 315:1575-8 (1986)). In this study, eight of ten patients improved their serum alanine transaminase (ALT) levels within one month of starting therapy. IFA therapy consisted of 5 million units (MU) daily in seven of the patients and one MU daily in three patients. In all subjects the dose was gradually reduced to 1 MU daily and then finally switched to an alternate day or every three day regimen. In three patients who had post-treatment liver biopsies, the specimen showed a marked improvement in the degree of portal inflammation and loss of parenchymal hepatocytic necrosis. Side effects were common at the 5 MU/day dose and virtually absent at 1 MU/day.
The effects of recombinant human interferon .alpha. in a prospective, randomized, double-blind, placebo-controlled trial in patients with well-documented chronic HCV infection has recently been carried out. Di Bisceglie, A. M., et al., New England Journal of Medicine, 321:1506-10 (1989). Forty-one patients were enrolled in the trial, 37 of whom were later found to have antibody to HCV. Twenty-one patients received interferon .alpha. (2 MU) subcutaneously three times weekly for six months, and twenty received placebo. The mean serum ALT and the histological features of the liver improved significantly in the patients treated with interferon, but not in the patients given placebo. Ten patients treated with interferon (48%) had a complete response, defined as a decline in mean serum ALT to the normal range during therapy; three others had a decrease in mean ALT of more than 50%. After treatment ended, however, serum ALT usually returned to pretreatment levels; six to twelve months after the discontinuation of interferon therapy, only two patients (10%) still had normal values. The authors concluded that interferon .alpha. therapy is beneficial in reducing disease activity in chronic hepatitis C; however, the beneficial responses are often transient and side effects are known to appear.
In another, broader study, chronic hepatitis C (NANB hepatitis) in 166 patients was treated with either 3 MU or 1 MU of recombinant human .alpha.-IFA three times weekly for 24 weeks or to no treatment. The serum ALT level became completely normal in 22 of the 26 patients (85%) who responded to treatment with 3 MU of interferon, and nine of the sixteen patients (56%) responded to treatment with 1 MU. The patients who received 3 MU of interferon had histologic improvement because of the regression of lobular and periportal inflammation. However, relapse within six months after the completion of treatment occurred in 51% of the patients treated with 3 MU of interferon and in 44% of those treated with 1 MU. Davis, G. L., et al., New England Journal of Medicine, 321:1501-06 (1989). These authors concluded that a 24-week course of interferon therapy is effective in controlling disease activity in many patients with hepatitis C, although relapse after the cessation of treatment is common.
A multi-center randomized control trial of recombinant human .alpha.-IFN in patients with chronic NANB hepatitis has been reported recently. Marcellin, P., et al., Hepatology, 13:393-97 (1991). Patients were randomly assigned to no treatment or to 1 to 3 MU of .alpha.-interferon given three times a week for 24 weeks. Forty-five patients (75%) were positive for antibody to HCV. During the 24 week treatment period, mean serum ALT levels decreased in both treatment groups, but the decrease was statistically significant only in the 3 MU group. However, at 24 weeks, the proportion of patients with normal ALT levels was similar in the 3 MU group (39%) and the 1 MU group (45%) and both were significantly higher than in controls (0%). Repeat liver biopsy specimens showed a significant decrease in the severity of histological changes in the higher dose group but not in the lower dose group or in controls. However, after treatment, the mean ALT levels rose in both treated groups. The proportion of patients with normal ALT levels at week 48 was 28% in the 3 MU group and 20% in the 1 MU group. The authors conclude that a dose of 3 MU was superior to 1 MU of .alpha.-interferon given three times per week for 24 weeks in inducing improvements in serum ALT levels and liver histological examinations. However, relapse in disease activity occurred in approximately half of the responders when interferon was stopped. The response to .alpha.-interferon did not correlate with the source of infection or with the presence or absence of anti-HCV antibody titres in patient sera.
It is clear, therefore, that while a-interferon has a beneficial effect on the course of HCV infection, this effect is frequently only transient. Therefore, new modalities are necessary in order permanently to eradicate the effects of hepatitis C virus on the patient.
Another class of polypeptide immune modifiers derived from the thymus gland, the thymosins, has been shown to trigger maturational events in lymphocytes, to augment T-cell function and to promote reconstitution of immune defects. Low, T. L. K., et al., "Thymosins: Structure, Function and Therapeutic Application", Thymus, 6:27-42 (1984).
Thymosin Fraction Five (TF-5), originally described by Goldstein et al. (Proc. Nat'l Acad. Sci. (USA), 69:1800-1803 (1972)), is a partially purified extract of bovine thymus containing at least 40 peptide components, 20 of which have been purified to homogeneity or near homogeneity; it contains about 0.6% of Thymosin .alpha.-1 (THN.alpha..sub.1). Low, 1984, above.
THN.alpha..sub.1, initially isolated from TF-5, has been sequenced and chemically synthesized. Wetzel, R., et al., Biochemistry, 19:6096-6104 (1980). Its sequence is highly homologous in mice, calves and humans. THN.alpha..sub.1 is a 28 amino acidic polypeptide with a molecular weight of 3100 that has shown activity qualitatively similar to TF-5 in modulating the immune system. Low, T. L. K., et al., J. Biol. Chem., 254:981-6 (1979). THN.alpha..sub.1 has potent immunologic activity, including stimulation of .alpha.- and .gamma.-interferon production, increasing macrophage migration inhibitory factor production, inducing expression of T-cell markers, including IL-2 receptors, and improving T-cell helper cell activity. Schulof, R. S., et al., in The Lymphocyte, Allen J. Liss Inc., New York, 1981, pp. 191-215; Low, T. L. K., et al., in "Thymosins: Structure, Function and Therapeutic Applications", Thymus, 6:27-43 (1984); Koutab, N. M., et al., Immunopharm., 16:97-105 (1988). Studies in mice have demonstrated a synergistic effect of THN.alpha..sub.1 and interferon on natural killer-cell activity in immunosuppressed mice. Favilli, C., et al., Cancer Immunol. Immunother., 20:189-92 (1985). TF-5 and THN.alpha..sub.1 can influence immunoregulatory T-cell function, promote production of interferon-.alpha., interferon-.gamma. and interleukin-2 by human lymphocytes and increase interleukin-2 receptor expression. Marshall, G. D., et al., J. Immunol., 126:741-4 (1981); Mutchnick, M. G., et al., Clin. Immunol. Immunopathol., 23:626-33 (1982); Sztein, M. B., et al., Proc. Nat'l Acad. Sci. (USA), 83:6107-6111 (1986); Serrate, S. A., et al., J. Immunol., 1939:2338-43 (1987); Baxevanis, C. N., et al., Immunopharm., 13:133-41 (1987); and, Svedersky, L. P., Eur. J. Immunol., 12:244-7 (1982).
Clinical trials of TF-5 and THN.alpha..sub.1 as primary or adjunctive therapy in patients with immunodeficiency or cancer indicate that these agents enhance immune responsiveness and augment specific lymphocyte functions. Clinical trials of TF-5 and purified THN.alpha..sub.1 have been underway for a number of years. Early trials in patients with cancer or immunodeficiency states were encouraging, though not definitive. Goldstein, A. L., et al., Transp. Proc., 9:1141 (1977); Barrett, D. J., et al., J. Pediatr., 97:61 (1980); and Cohen, M. H., et al., J. Amer. Med. Assoc., 241:1813-5 (1979). THN.alpha..sub.1 use has been described in a randomized trial of patients with nonsmall cell lung cancer. Patients were treated with THN.alpha..sub.1 at a dose of 900 .mu.grams/m.sup.2 subcutaneously twice weekly or daily for two weeks and then twice weekly after completing a course of radiotherapy. The only side effect of THN.alpha..sub.1 was mild burning at the injection site in three patients. This was attributed to the drug lot and may have been due to the carrier preparation. Relapse-free survival and overall survival were greater in both THN.alpha..sub.1 treatment groups than in the placebo group; some restoration of radiation-suppressed immune function was also noticed. There was no increase in T-cell numbers associated with this. Schulof, R. S., et al. J. Biol. Response Modifiers, 4:147-58 (1985).
Recent double-blind, randomized trials with thymosins have been performed in elderly men in an effort to increase response to influenza vaccine. Gravenstein, S., et al., JAGS, 37:1-8 (1989). Patients received synthetic THN.alpha..sub.1 subcutaneously twice weekly starting at the time the influenza vaccine was given. At six weeks post-vaccine, those patients randomized to receive the drug had higher levels of antibody to influenza than controls. This difference was accentuated in the very elderly (ages 77-99). No clinical or biochemical toxicity was observed in drug recipients.
There are preliminary reports that thymosins may be effective against infections caused by hepatitis viruses other than HCV. In an animal model of viral hepatitis, the woodchuck infected with the Woodchuck Hepatitis Virus, THN.alpha..sub.1 suppressed viral DNA replication, but produced no improvement in clinical parameters. Korba, B. E., et al., Hepatology, 12:Abs. 880 (1990). In a pilot clinical trial with patients with Chronic Active Hepatitis B caused by the hepatitis B virus (HBV), patients treated for a year with THN.alpha..sub.1 (5 patients) or with TF-5 (2 patients) showed a marked decrease in serum ALT; 6 of the 7 patients also showed reduced levels of serum HBV DNA, and 5 of 6 patients initially positive for serum hepatitis B surface antigen (HBSAg) subsequently cleared this antigen. Mutchnick, M. C., et al., Hematology, 10:Abs. 575 (1989). No suggestion was made in these abstracts that the thymosins would be effective against any other hepatitis viruses.
There remains, therefore, an important need in the art for a new modality for the treatment of HCV infections in mammals; this modality is disclosed below.